Histidine triad (HIT) proteins are a superfamily of nucleotide hydrolases consisting of two branches we have characterized structurally, biochemically and genetically. Mammalian Hint and yeast homolog Hnt1 hydrolyze the natural product AMPNH2 and we showed that enzyme activity is required for biological function. Yeast hnt1 mutants fail to grow at elevated temperatures on galactose and are synthetically less viable with mutants in TFIIK components Kin28, Ccl1 and Tfb3, and with Cak1. The Fhit branch of the HIT superfamily contains enzymes that hydrolyze diadenosine polyphosphates (ApnA). Deletions in human FHIT are among the earliest and most frequent genetic changes in epithelial tumors such as lung, which are responsible for 100,000s of annual US deaths. While yeast genetic experiments prove that Fhit homolog, Hnt2, controls ApnA levels in vivo, specific ablation of the ability of Fhit to hydrolyze but not to bind ApnA does not block the ability of Fhit to suppress tumor formation. These discoveries contributed to a Ras-like model of Fhit function in which the crystallographically defined Fhit-substrate analog complex is seen as the active signaling form. The structure led to novel fluorescent substrates, aided inhibitor synthesis and characterization, and wild-type and mutant enzymology, and began to enable Fhit imaging/diagnostics. Nonetheless, the cellular mechanism of Fhit as tumor suppressor and cellular function of Hnt2 are major unsolved problems that are tractable via synthetic lethal analysis. Specific aims of this proposal are as follows. 1) We will use chemical methods to define the small molecule and/or polypeptide Hnt1 substrates that account for genetic interactions between kin28 and hnt1 2) We will apply biochemical and genetic methods to discover the mechanism for the gene expression and carbon source utilization consequences of hnt1-deficiency. 3) We will complete a synthetic lethal and suppression screen with hnt2 and probe a protein array with APnA-bound Hnt2 and use these genes to characterize HNT2 function. 4) We will use a collection of Fhit mutants we have purified to test rigorously the hypothesis that Fhit-substrate complexes are the active signaling form of Fhit. Work on Hint/Hnt1 will clarify a newly discovered, conserved regulatory mechanism acting on TFIIK that affects carbon source use and cell viability in yeast, which appears related to the defect responsible for ataxia with oculomotor apraxia. Work on Fhit/Hnt2 is designed to uncover the pathway of one of the most frequently lost cancer genes and may lead to identification of novel targets for prevention and treatment of lung and stomach cancer.